Hit identification


Evotec has a strong expertise in hit identification and can support its clients screening campaigns through multiple platforms including virtual, biochemical, biophysical, cell and phenotypic based screening with high-throughput capabilities.

For screening campaigns, Evotec has the flexibility to screen large client compound collections, its own 450,000 Evotec and 400,000 Aptuit compounds collection. 

Evotec state-of-the-art platforms offer a large variety of detection technologies, assay development and miniaturisation capabilities and the use of multiple read-out parameters to enable the reduction of false positives and negatives. In addition, Evotec offers to review hit compounds from a biological and medicinal chemistry point of views to ensure that compounds further progressed have the required drug like profiles.

Through an understanding of each client’s project requirements and goals, our experienced screening team will design the most adequate hit identification campaign ensuring the highest chance of success.

Screening Collections

The Aptuit collection

Aptuit’s library contains 400,000 compounds which are complementary to the Evotec collection. It is consisting of 350,000 maximally diverse lead-like “islands” with low lipophilicity plus a 50,000 “ligand efficiency” collection.

Distribution of calculated properties in Evotec`s discovery screening library

The Evotec collection

Evotec’s library contains 450,000 compounds consisting of 30,000 proprietary Evotec compounds, a set of 370,000 maximally diverse “islands” with lead-like properties, 20,000 fragments, 5,000 compounds with known bio-annotation and 25,000 natural products, macrocycles and natural product derivatives from the Analyticon collection.

Evotec’s screening library is differentiated through its quality, diversity and novelty and has a successful track record of delivering potent and attractive compounds as starting points for medicinal chemistry optimisation.

For structure-based approaches, Evotec has a 20,000 fragment set available for biochemical screening, plus 3,000 (Evotec) and 1,500 (Aptuit) fragment sets specifically selected for NMR and SPR screening and finally a 330 compound collection for crystal soaking.


Third party and other collections

Evotec can also screen customer’s libraries of any size, virtual screening derived hits and also smaller, more target directed libraries.

High-Throughput Screening

Evotec has a long history in high-throughput screening utilising multiple compound collections and has run more than 320 cell based, micro-organism based or biochemical HTS campaigns. Leveraging this expertise, a state-of-the-art screening platform, a strong experience in assay development and miniaturisation (>475 assays developed), and an excellent disease biology knowledge, Evotec selects the most suitable screening strategy for your target.

Compound screening process

The integration and connection of the different stages of the screening process from assay development, automation, compound management and tracking to data acquisition and processing are essential. The Evotec screening team manages this complexity on a daily basis to deliver high-quality data to its clients in the most flexible manner to meet customers’ needs in data format for upload in different environment.

Assay systems

Evotec has twenty years of experience in assay development; in particular in HTS, but also to support hit-to-lead and lead optimisation (H2L/LO) programmes.

>350 uHTS screens with sSpecific hits with EC50<25µM identified in most screens

We believe that the success of a screening project is determined by: 
  • The target selection
  • The right strategy for target expression to address specific sites of interest
  • A robust and sensitive assay system
  • A well-selected compound collection for screening
  • A reliable screening process including counter and orthogonal assays
  • A careful characterisation of screening hits by MedChem assessment
  • A continuous interaction with our clients on the progress of a screening project

>475 assays developed, success rate >95%, high percentage of cellular assays

Our assay system capabilities and experience include:
  • A broad portfolio of assays addressing the major therapeutically relevant target classes: our team has developed more than 475 assays to support drug discovery programmes
  • Assay miniaturisation for over 350 cell based, micro-organism based or biochemical HTS campaigns
  • New target classes in the field of epigenetics, transporter biology and protein-protein interaction
  • Anti-infective screens ran on multiple pathogenic strains
  • High numbers of unique assays developed as no literature precedence was available
  • Assay read-outs covering a wide range of technologies including biochemical, molecular and cellular assays
  • Assays using label-free and biophysical methods

HTS platforms

Evotec’s high-throughput screening platform is differentiated through integrating a large variety of detection technologies to state-of-the-art high-throughput capabilities.

  • Variety of automation systems available to offer high flexibility with regard to assay plate formats, assay read-outs, readers and dispenser/washers
  • Overall capacity of running up to 45 HTS campaigns per year
  • Maximum throughput of 1536-well plate systems up to 100,000 data points per system/day
  • Fully automated support of RapidFire/MS screening campaigns (up to 10,000 data points per system/day)
  • Infrastructure and permissions in place to run radiometric and biosafety level 2 screening campaigns

HT screening of micro-organisms

Evotec has a a very strong background in anti-infective disease; leveraging an exceptional  in vitro and in vivo biology knowledge in anti-infectives and a comprehensive portfolio of assays, our team provides full bio-organisms screening capabilities against Biosafety Class 2 biological agents.

HT screening of micro-organisms

Micro-organism screening capabilities include:
  • Recombinant human cells or primary cells
  • Micro-organisms: bacteria, viruses and yeast & fungi
Read-out technologies include:
  • Absorbance
  • Luminescence
  • Fluorescence
  • HTRF
Screening assay principles in 384-well and 1536-well plate formats include:
  • Phenotypic assays on growth inhibition of micro-organisms and cells
  • Target-based assays: biochemical assays, cellular assays with gene reporter and cell-based assays with the incubation of cells and pathogen

RT-qPCR screening

Evotec´s assay development and HTS track record includes high-throughput RT-qPCR capabilities. This technology allows to investigate cellular mRNA fluctuation in response to treatment with small molecule compounds, foreign RNA and protein therapeutic candidates.

RT-qPCR screening

RT-qPCR screening capabilities and expertise include:
  • A high-throughput approach to gene expression profiling with RT-qPCR as a primary screening tool
  • Established screening processes for 384- and 1536-well plate assays 
  • Automated screening system consists of incubators, washer/dispensers, nanoliter Echo550 acoustic liquid handler, real time PCR 1536 LightCycler
  • Throughput of 10 up to 20 plates/day in 384 well plate format and up to 20 plates/day in 1536-well plate format

High-Content Screening

Evotec has strong expertise in the development and implementation of high-content based assays for high-throughput screening and compound validation to probe the pathophysiological  environment.
High-content screening (“HCS”) is routinely used throughout the hit identification cascade from target validation to mechanistic studies. The assays developed at Evotec encompass both a target centric as well as phenotypic approach with emphasis on tissue samples, stem cell derived or primary cells from appropriate hosts. This approach is a core part of Evotec’s efforts to identify novel targets and drugs with disease-modifying potential.

Opera Phenix™

Evotec’s HCS expertise is built around the OPERA® platform where an essential understanding of the mechanics and image analysis tools, gathered over a period of more than 15 years in the development of the platform, flows into the hit identification process. Additional platforms include the Operetta® and ArrayScan™ to fulfill different requirements in resolution and throughput. The platform is completed with a powerful data management system and powerful data analysis tools including multifactorial analysis.
Based on this expertise, Evotec has performed many high-throughput screens of up to 400,000 compounds and developed over 30 assays to study mode of action of compounds and targets in a multitude of cellular backgrounds.


  • Proliferation
  • Cell cycle analysis
  • Cell death/necrosis/apoptosis
  • Differentiation markers
  • DNA damage
  • Cytoskeleton rearrangements
  • Neurite outgrowth
  • Mitochondrial mass/-toxicity
  • Endocytosis
  • Synapse density
  • Neurodegeneration/-protection
  • Intracellular aggregation
  • Post-translational modification

Structural Biology and Biophysics

Evotec has a comprehensive structural biology and biophysics platform. Backed by years of experience the team will design the optimal experiment to meet the demands of research budgets and timelines. The platform delivers binding information from atomic resolution of binding pose through to residency times and the kinetic profiling of target engagement. The information provided empower research programmes to accelerate their discovery efforts through rationale, hypothesis-driven design.

NMR spectrometer

The platform for structural biology and biophysics includes the following techniques:
  • Macromolecular crystallography
  • NMR
  • Electron microscopy
  • SPR
  • DSF
  • Affinity mass spectroscopy
  • MST
  • ITC

The established work flow includes a complete fragment-based drug discovery (“FBDD”) platform. Sensitive screening techniques, such as the FCS+plus (proprietary to Evotec) or NMR, SPR,  DSF-mass spectrometry (MS) or Crystal Soaking, combined with specifically selected fragment compound collections, identify diverse, active low molecular weight fragments providing multiple highly efficient starting points for chemical elaboration.  Routine and high-throughput crystallography of the fragment target complex delivers efficient fragment evolution, driving optimisation of potency and selectivity.

Biophysics – Screening using labelled and non-labelled methods

NMR screening and hit qualification

NMR screening as part of the Evotec fragment based design platform (FBDD) platform is an option for the identification of new starting points for structure based drug discovery (SBDD) programmes. Evotec offers both protein-observed NMR assays monitoring chemical shift perturbations (SAR-by-NMR) as well as ligand-detected NMR assays (Saturation Transfer Difference “STD” NMR and Water LOGSY) for hit identification. Hence, a broad range of target proteins amenable to NMR screening technique can be covered.
Evotec has employed its NMR screening platform on more than 30 protein targets covering a whole range of target classes from frequently studied enzymes (like kinases or proteases) over nucleotide binding proteins or epigenetic targets, to protein-protein interaction targets, even receptor domains.
NMR also adds considerable value to biochemical screening campaigns by validating hit compounds for direct binding to the target protein and ultimately correlating this information to structure data, thereby enabling or facilitating SBDD programmes.

Surface plasmon resonance (“SPR”) screening

SPR technology represents a powerful tool for studying biomolecular interactions in a sensitive and label-free manner in real-time.

SPR  screening

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Biacore™ 8k

Biacore™ 8k

Binding of maltose to immobilised MBP

Binding of maltose to immobilised MBP

Concentration response curve for maltose binding to immobilised MBP

Concentration response curve for maltose binding to immobilised MBP

While commonly used at later stages of drug discovery projects to qualify hit compounds with regards to binding kinetics, the SPR technology available at Evotec also allows for primary screening of fragment libraries.
Direct binding assays are run on the Biacore™ 4000 or Biacore™ 8k screening devices enabling the identification of fragments interacting with immobilised target proteins. As the throughput is sufficiently high, we are able to screen large fragment sets thus increasing the chance to find bona fide binders of the target.

Proprietary high-concentration bioassay (FCS+plus )

Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuation of fluorescence intensity. Applying this technology, a very tiny space in a microtitre plate well (confocal volume; less than 1 femtolitre), containing a small number of fluorescent particles, is illuminated by a laser. The fluorescence emitted from this confocal volume is analysed. The analysis reveals e.g. the average number and hence, the concentration of the fluorescent particles, average diffusion time through the confocal volume, fluorescence intensity, molecular fluorescent brightness, fluorescence lifetime, translational and rotational diffusion of the fluorescent particles. This technology is highly sensitive and allows to detect single molecules in solution and to distinguish different molecular species within a mixture of fluorescently labeled species; e.g. detection of ligand and respective receptor/ligand complex or detection of substrate and product of an enzymatic reaction at the same time.

Mass Spectrometry (MS/MS)

Evotec offers affinity screening by SEC-LC/MS and enzymatic screening by SPE-MS/MS:

  • Affinity binding: Through a rapid assay development, this label free technology allows to test all possible binding site at once. Mixture of compounds (up to 200 compounds per well) can be screened, hit confirmation is performed with pure compound. Up to 20,000 compounds (in mixture) can be screened per day
  • Enzymatic screening: This label-free technology allow a direct read-out of enzymatic reactions in biochemical or cellular assay via MS detection of substrate, products. The fully automated process allows screening of up to 7,000 samples per day and system

Crystallography screening

Offering a complete gene to structure package we deliver a complete crystal soaking fragment screening service.  The diverse Evotec Fragments-for-Crystals (FragTal) collection, chosen by our in-house computational chemistry team, can be screened against any suitable crystal structures. Delivering fragment hits to launch discovery chemistry programmes.  The fragment collection can be expanded as necessary to focus on a target or augmented by collaborator’s fragment collection. Streamlined work flows are established from sample preparation through to data analysis.

Isothermal Titration Calorimetry ("ITC"), and Microscale Thermophoresis (“MST”) and Differential Scanning Fluorimetry “DSF”  (Thermal shift) 

We typically combine SPR or NMR approaches with orthogonal assays and technologies like Isothermal Titration Calorimetry (“ITC”), Differential Scanning Fluorimetry (“DSF”) and Microscale Thermophoresis (“MST”).

ITC is a label-free, in solution technique considered to be the “gold standard” for measuring a broad range of ligand interactions. It allows the full thermodynamic dissection of an interaction (Kd, stoichiometry) delivering details unavailable through other techniques.


MST and DSF examples

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Thermophoretic diffusion of MBP in presence of increasing concentrations of maltose

Thermophoretic diffusion of MBP in presence of increasing concentrations of maltose

Concentration response curve for maltose binding to MBP

Concentration response curve for maltose binding to MBP

Thermal unfolding of MBP in presence of increasing concentrations of maltose

Thermal unfolding of MBP in presence of increasing concentrations of maltose

Concentration dependent stabilisation of MBP by maltose

Concentration dependent stabilisation of MBP by maltose

MST is applied in two modes available, the label free as well as the labelled version of the technology. This allows for maximal flexibility on a broad variety of targets in conjunction with low affinity fragment like compounds. Through a fast assay development and a fast Kd determination, binding affinities of proteins, nucleic acids, fragments, peptides, small molecules with non-immobilized proteins can be evaluated.

DSF provides binding affinity information of proteins, nucleic acids, fragments, peptides, small molecules through stability measurement. This technology does not require immobilisation as interaction can be evaluated in solution. Via a fast assay development, up to 30,000 compounds per day can be analysed with a moderate protein consumption. 

Structural biology - Bio reagents


Evotec offers rapid production of high quality recombinant proteins for structural studies. Working with the comprehensive panel of protein expression systems, we generate protein reagents to meet collaborator needs. The structural biology unit, with many years of experience across all target classes, routinely produces proteins for structural studies.

Bio-reagents offers full gene-to-protein services:

  • Construct design 
  • Expression in E. coli, yeast, mammalian or baculovirus/insect cells and cell free 
  • Fermentation and wave bags to 100 L scale
  • Automated protein purification and protein refolding 
  • Purity of >95% routinely achieved and comprehensive QC 
  • Labelled proteins for NMR

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Bio reagents

Macromolecular crystallography and structural biology

Being located within a few miles of the UK’s third generation synchrotron and leveraging its own in-house X-ray source, Evotec offers the ideal infrastructure for rapid cycle times in X-ray crystallography.
The Structural biology team provides solutions to novel structure elucidation of drug targets and optimisation of crystal systems to support industrialisation of protein:ligand complex elucidation.
The team has co-developed with Diamond Light Source a high-throughput fragment screening by X-ray crystallography. Evotec has a 3d-enriched fragment library and has demonstrated repeatedly within a matter of weeks the delivery of immediate structures to stimulate medicinal chemistry campaigns.

Guided by highest-quality structural data, SBDD at Evotec distils information into hypothesis-driven medicinal chemistry. By minimising unnecessary and wasteful compound synthesis, productivity is increased and chemistry teams are able to focus on an efficient path to the final drug.

The structural biology platform includes:

  • Access to a team of 12 dedicated PhD level structural biologist with >80 years combined industrial experience across a diverse range of target proteins, antibodies, protein complexes and protein:nucleic acid complexes to support client needs
  • Co-localisation with extended protein production team to ensure the best quality protein is delivered immediately into structural studies
  • Development of de novo and literature crystal systems suitable for the demands of industrial crystallography
  • Fast cycle time routine support by crystallography for SBDD with regular and routine access to synchrotron sources across Europe and the USA
  • Access to high efficiency synchrotron-based X-ray fragment screening
  • bioSAXS to address questions of in-solution conformational change
  • Circular dichroism to monitor secondary structure changes
  • Cryo-Electron microscopy and other related techniques using the latest microscopes through access to eBIC (electron Bio-Imaging Centre) the UK’s national electron microscopy facility hosted locally at Diamond Light Source

Virtual Screening and hit expansion

As part of its large portfolio of screening capabilities, Evotec offers virtual screening and hit expansion campaigns to its customers.

Virtual screening

Leveraging a strong expertise in research informatics and in silico design, our scientists will design the most suitable in silico screening campaign to meet our clients’ requirements.
For virtual screening, Evotec has access to multiple methods using the best of-breed commercial software.

The screening campaign can be run using number of compound collections:
  • EVOspace
  • EvoSource (34 million compounds)
  • Evotec diverse compound collection (400,000 compounds)
  • Evotec fragment library (20,000 compounds)
  • Evotec diverse phenotypic library (20,000 compounds)
  • An annotated druglike library (5,000 compounds)

Chemogenomics and ortholog approaches are also available to our client and provide a good starting point for identifying novel active compounds.
Evotec has licensed LeadIT and Spark for scaffold hopping but also has a number of in-house tools using structure merging and vector placement algorithms.

Hit expansion

The hits identified from screening require expansion to provide evidence of SAR. This is traditionally based upon chemical similarity and involves searching databases for ‘like’ molecules. Evotec also uses predictive pharmacology so the molecules that possess a similar target pathway signature can be identified.
Library design, statistical analysis and virtual chemistry tools can be used to design the most information rich set of molecules for synthesis.

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